Homo- and heterodimeric protein complexes mediate many cellular processes and abnormal protein interactions underly various medical conditions. Yan et al. (1995) Cancer-Res. 55: 3569-75. Research on such complexes has led to efforts to understand disease at the molecular level and to a search for small molecule effectors of such complexes. Such effectors could modulate protein interactions and are potential therapeutic agents. Gibbs & Oliff (1994) Cell 79: 193-198. Most often, such effectors have been identified using various biochemical and immunological in vitro approaches. The advantages of genetic approaches in drug discovery, however, have recently received increased attention. Liuzzi et al. (1994), Nature 372: 695-8. These advantages include both cost-effectiveness and simplicity. Only one such genetic system, the yeast-two hybrid system, currently meets all these criteria and is also equally suitable for the detection of both homo- and heterodimeric protein interactions. Another unique feature of the yeast two-hybrid system is its ability to detect the desired protein-protein interaction without interference by competing interactions. Fields & Song (1989) Nature 340: 245-6. The system has been successfully used for the analysis of protein interactions and for the isolation of interacting proteins through interaction cloning. For a review, see Allen et al. (1995), Trends in Biochem. Sci. 20: 511-16.
Although the yeast two-hybrid system has proven highly useful, it suffers from a number of limitations. Yeast is impermeable to many small molecules, which effectively prevents their evaluation in a yeast system. Higgins (1993) Curr. Opin. in Cell Biol., 5: 684-687. The yeast system also requires nuclear localization of interacting proteins, which may lead to other complications.
These problems can potentially be overcome with an E. coli two-hybrid system. E. coli strains can be hyperpermeable. Nakamura & Suganuma (1972) J. Bacteriol. 110: 329-35. One can use this hyperpermeability to maximize the number of small molecules that can be evaluated. In addition, E. coli has a rapid growth rate, permitting shorter turnaround times during drug screening. Furthermore, one can transform E. coli at high frequencies, facilitating interaction cloning.
To date, only one E. coli system seems to have properties similar to the yeast two-hybrid system, but this system has only been shown to detect homodimerization of an E. coli protein and there is no published evidence that the system is sufficiently robust to be useful for major two-hybrid applications such as interaction cloning. Dove et al. (1997), Nature 386: 627-30. Other E. coli systems for detecting protein interactions genetically have also been reported. Doerr et al. (1991) Biochem. 30: 9657-64; Marchetti et al. (1995) J. Mol. Biol. 248: 541-50; Jappelli & Brenner (1996) J. Mol. Biol. 259: 575-8. Unlike the yeast two-hybrid system, however, these systems require homodimerization of at least one of the interacting proteins and detection of the desired protein-protein interaction can be subject to interference by competing other interactions. The art would benefit from an E. coli two-hybrid system that can detect a variety of protein interactions and which is sufficiently robust for interaction cloning.